Generation of HLA-DR+CD1c-CD14-XCR1+ dendritic cells expressing CAR from induced pluripotent cells as an alternative treatment for cancer

Abstract

Background & Aim

Immunotherapy based on T cells expressing CARs has been shown to be a promising strategy for treating hematological tumors. However, similar results have not been reported for solid tumors. Therefore, other immune cells have been explored to express CARs for immunotherapy applications. Dendritic cells are a heterogeneous cell population specialized in antigen presentation, with great migratory capacity, representing good candidates for solid tumor therapies. Due to their scarcity in peripheral blood and low proliferative potential, an "off-the-shelf" source such as induced pluripotent stem cells is needed to enable their use in cell therapy. Thus, our aim is to develop iPSC-DCs expressing CAR as an alternative treatment for solid tumors.

Methodology

Electroporation of the piggyBac transposon system was utilized to insert a CAR into an iPSC line. Differentiation of CAR-iPSCs was carried out following an established step-by-step protocol: (A) embryoid body formation, (B) generation of hematopoietic stem-like cells and (C) production of immune myeloid-like cells. Subsequently, differentiation into myeloid immune cells was induced using GM-CSF and IL-4 after 30 days. Flow cytometry was employed to analyze the phenotype of the differentiated cells. Co-culture of CAR-iPSC-DC cells with T cells was performed to observe induction of T cell proliferation.

Results

We have achieved an initial transfection efficiency of 2%. Sequential cell sorting increased the purity of CAR-expressing cells to 95%, with stable expression maintained for over 40 days. We obtained differentiated cells with typical myeloid immune cell morphology, including a few macrophage-like cells, with large cytoplasmic vacuoles and numerous cells with dendrite projections, closely resembling DC morphology. Moreover, cells presented a HLA-DR+CD64lowXCR1high phenotype, similar to cDC1 found in PB. Preliminary data showed that these cDC1-like cells are able to induce CD3+ T cell proliferation in vitro, similar to that observed with moDCs, reinforcing their similarity with PB DCs.

Conclusion

This study highlights the feasibility of using iPSC-derived DCs as an "off-the-shelf" cell source for cancer immunotherapies. The observed cDC1-like phenotype and the ability of these cells to stimulate CD3+ T cell proliferation in vitro provide a strong foundation for further investigation into their functionality and antitumor potential. The next steps include in vitro and in vivo analysis of cDC1-like function and their antitumor effects.